USOO6322895B1 (12) United States Patent (10) Patent No.: US 6,322,895 B1 Canham (45) Date of Patent: Nov. 27, 2001

(54) BIOMATERIAL 5,348,618 9/1994 Canham et al. . 5,358,600 * 10/1994 Canham. (75) Inventor: Leigh T Canham, Malvern (GB) FOREIGN PATENT DOCUMENTS (73) Assignee: QinetiQ Limited, London (GB) 361218932A * 9/1986 (JP). 405049691A * 3/1993 (JP). * Y NotOtice: Subjubject to anyy disclaimer,disclai theh term off thisthi 405337137A * 12/1993 (JP). patent is extended or adjusted under 35 6-169981 6/1994 (JP). U.S.C. 154(b) by 0 days. (21) Appl. No.: 09/000,258 OTHER PUBLICATIONS (22) PCT Filed: Aug. 1, 1996 Database WOPWeek 8645 Derwent Publications Ltd., Lon don, GB; AN 86-295840 XP002021586 & JP, A,61218932 (86) PCT No.: PCT/GB96/01863 (Toko), Sep. 29, 1986 see abstract. S371 Date: Jan. 30, 1998 Database WPI Week 9429 Derwent Publications Ltd., Lon don, GB; AN 94-237642 XO002021587& JPA,06 169981 S 102(e) Date: Jan. 30, 1998 (Erusoru), Jun. 21, 1994 see abstract. (87) PCT Pub. No.: WO97/06101 * cited by examiner PCT Pub. Date: Feb. 20, 1997 (30) Foreign Application Priority Data Primary Examiner Deborah Jones Assistant Examiner Stephen Stein Aug. 3, 1995 (GB) ...... 9515956 (74) Attorney, Agent, or Firm Nixon & Vanderhye Nov. 28, 1995 (GB) ...... 9524242 May 31, 1996 (GB) ...... 9611437 (57) ABSTRACT (51) Int. Cl." ...... B32B 9/00 Bioactive Silicon comprising a porous form of Silicon which (52) U.S. Cl...... 428/450; 428/446; 427/58 when in Vivo elicits a Specific biological response that (58) Field of Search ...... 428/446, 450; results in the formation of a bond between living tissue and 427/2.24, 2.27, 457, 58; 204/403 the Silicon. The deposition of apatite provides an indication that the porous Silicon is bioactive and therefore biocom (56) References Cited patible. Bioactive silicon may be used in the fabrication of biosensors for in vitro or in Vivo applications. U.S. PATENT DOCUMENTS 4,569,743 * 2/1986 Bayer et al...... 204/192.5 6 Claims, 7 Drawing Sheets

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N VO s (Y V ve 8 2 9 9 O 92 O 9 9 SNO) NO WONOOS US 6,322,895 B1 1 2 BOMATERIAL al. in the Journal of Materials Science: Materials In Medicine, Volume 1, 1990, pages 233 to 238. This is a 35 U.S.C. S371 of PCT/GB96/01863, filed Aug. There is a long felt want for the ability to use silicon 1, 1996. based integrated circuits within the human body both for The present invention relates to biomaterials. diagnostic and therapeutic purposes. Silicon has been A"biomaterial' is a non-living material used in a medical reported to exhibit a poor biocompatibility in blood, Kanda device which is intended to interact with biological Systems. et al. in Electronics Letters, Volume 17, Number 16, 1981, Such materials may be relatively “bioinert”, pages 558 and 559, and in order to protect integrated circuits “biocompatible”, “bioactive” or “uresorbable', depending from damage in biological environments encapsulation by a on their biological response in Vivo. Suitable material is currently required. Medical applications Bioactive materials are a class of materials each of which for Silicon based Sensors are described in a paper by Engels when in Vivo elicits a Specific biological response that et al. in the Journal of Physics E.: Sci. Instrum., Volume 16, results in the formation of a bond between living tissue and 1983, pages 987 to 994. that material. Bioactive materials are also referred to as The present invention provides bioactive Silicon charac Surface reactive biomaterials. Biomaterials may be defined 15 terized in that the Silicon is at least partly crystalline. as materials Suitable for implantation into a living organism. Bioactive Silicon provides the advantage over other bio L. L. Hench has reviewed biomaterials in a Scientific paper active materials that it is compatible with Silicon based published in Science, Volume 208, May 1980, pages integrated circuit technology. It has the advantage over 826-831. Biomaterials which are relatively inert may cause non-bioactive Silicon that it exhibits a greater degree of interfacial problems when implanted and So considerable biocompatibility. In addition, bioactive Silicon may be used research activity has been directed towards developing for forming a bond to bone or vascular tissue of a living materials which are bioactive in order to improve the animal. Bioactive Silicon may provide a material Suitable for biomaterial-tissue interface. use as a packaging material in miniaturised packaging Known bioactive materials include hydroxyapatite (HA), applications. Some glasses and Some glass ceramics. Both bioactive 25 The bioactive nature of the silicon may be demonstrated glasses and bioactive glass ceramics form a biologically by the immersion of the material in a simulated body fluid active layer of hydroxycarbonateapatite (HCA) when held at a physiological temperature, Such immersion pro implanted. This layer is equivalent chemically and structur ducing a mineral deposit on the bioactive Silicon. The ally to the mineral phase in bone and is responsible for the mineral deposit may be apatite. The apatite deposit may be interfacial bonding between bone and the bioactive material. continuous over an area greater than 100 um. The bioactive The properties of these bioactive materials are described by Silicon may be at least partially porous Silicon. The porous L. L. Hench in the Journal of the American Ceramic Society, Silicon may have a porosity greater than 4% and less than Volume 74 Number 7, 1991, pages 1487-1510. The scien 70%. tific literature on bioactive materials often uses the terms HA Bulk crystalline Silicon can be rendered porous by partial and HCA on an interchangeable basis. In this patent 35 electrochemical dissolution in hydrofluoric acid based specification, the materials HA and HCA are collectively Solutions, as described in U.S. Pat. No. 5,348,618. This referred to as apatite. etching process generates a Silicon Structure that retains the Li et al. have reported the deposition of apatite on Silica crystallinity and the crystallographic orientation of the origi gel in the Journal of Biomedical Materials Research, Volume nal bulk material. The porous silicon thus formed is a form 28, 1994, pages 7-15. They Suggest that a certain density of 40 of crystalline Silicon. At low levels of porosity, for example Silanol (SiOH) groups is necessary to trigger the heteroge less than 20%, the electronic properties of the porous Silicon neous nucleation of hydroxyapatite. An apatite layer did not resemble those of bulk crystalline silicon. develop on the Surface of a Silica glass Sample and this is Porous Silicon may be Subdivided according to the nature attributed to the lower density of Surface Silanol groups of the porosity. Microporous Silicon contains pores having a compared with Silica gel. 45 diameter less than 20 A; mesoporous Silicon contains pores Thick films of apatite have previously been deposited on having a diameter in the range 20 A to 500 A; and Silicon Single crystal wafers by placing the wafers in close macroporous Silicon contains pores having a diameter proximity to a plate of apatite and Wollastonite-containing greater than 500 A. The bioactive silicon may comprise glass dipped into a physiological Solution at 36 C., as porous Silicon which is either microporous or mesoporous. described by Wang et al. in the Joumal of Materials Science: 50 Silicon has never been judged a promising biomaterial, Materials In Medicine, Volume 6, 1995, pages 94-104. A in contrast with numerous metals, ceramicS and polymers, physiological Solution, also known as a simulated body fluid and has never been judged capable of exhibiting bioactive (SBF), is a Solution containing ion concentrations similar to behavior. Indeed, no Semiconductors have been reported to those found in the human body and is widely used to mimic be bioactive. Silicon is at best reported to be relatively the behavior of the body in vitro tests of bioactivity. Wang 55 bioinert but generally exhibits poor biocompatibility. et al. reported the growth of apatite on (111) Si wafers but Despite the advances made in miniaturisation of integrated reported that “hardly any” apatite could be grown on (100) circuitry, silicon VLSI technology is still under development Si wafers. The silicon wafer itself is not bioactive. Wang et for invasive medical and biosensing applications, as al. State that "Si does not play any special role in the growth described by K. D. Wise et al. in “VLSI in Medicine” edited of (the) apatite film except that Siatoms on the Substrate can 60 by N. G. Einspruch et al., Academic Press, New York, 1989, bond Strongly with oxygen atoms in apatite nuclei to form Chapter 10 and M. Madou et al. in Appl. Biochem. interfaces with low energy'. The presence of the apatite and Biotechn., Volume 41, 1993, pages 109-128. Wollastonite containing glass is required to induce the depo The use of Siliconstructures for biological applications is Sition of the apatite. Indeed, this So-called “biomimetic known. International patent application PCT/US95/02752 process” whereby a bioactive material is used to treat 65 having an International Publication Number WO95/24472 another material has been shown to induce apatite growth on describes a capsule having end faces formed from a perfo a wide variety of bioinert materials, as reported by Y. Abe et rated amorphous Silicon Structure, whose pores are large US 6,322,895 B1 3 4 enough to allow desired molecular products through but In a further aspect, the invention provides a bioactive which block the passage of larger immunological molecules, Silicon Structure characterized in that the Silicon is at least to provide immunological isolation of cells contained partly crystalline. therein. No evidence as to the biocompatibility of the silicon In a still further aspect, the invention provides an elec structure is provided, and workers skilled in the field of tronic device for operation within a living human or animal biocompatible materials would expect that Such a device body, characterized in that the device includes bioactive would in vivo stimulate the production of fibrous tissue Silicon. which would block the pores. It is known that when micro Bioactive Silicon of the invention may be arranged as a machined Silicon Structures are used as Sensors for neural protective covering for an electronic circuit as well as a means for attaching a device to bone or other tissue. elements a layer of fibrous tissue forms between the silicon The electronic device may be a Sensor device or a device Surfaces and the neural elements of interest, as reported by for intelligent drug delivery or a prosthetic device. D. J. Edell et al. in IEEE Transactions on Biomedical In a still further aspect, the invention provides a method Engineering, Volume 39, Number 6, 1992 page 635. Indeed of making Silicon bioactive wherein the method comprises the thickneSS and nature of any fibrous tissue layer formed making at least part of the Silicon porous. is often used as one measure of biocompatibility, with a 15 In another aspect, the invention provides a method of thinner layer containing little cell necrosis reflecting a higher fabricating bioactive Silicon, characterized in that the degree of biocompatibility. method comprises the Step of depositing a layer of poly U.S. Pat. No. 5,225,374 describes the use of porous crystalline Silicon. Silicon as a Substrate for a protein-lipid film which interacts In a yet further aspect, the invention provides biocom with target Species to produce an electrical current when patible Silicon characterized in that the Silicon is at least exposed to target Species in an in vitro Solution. The porous partly crystalline. Silicon is oxidised to produce a hydrophilic Surface and is In a still further aspect, the invention provides resorbable chosen Since the pores act as a conduit for an ion-current Silicon. flow and the Structure provides Structural Support for the In another aspect, the invention provides a method of lipid layer. The porous Silicon is separated from the in Vitro 25 accelerating or retarding the rate of deposition of a mineral Solution by the protein-lipid film and So the question of the deposit on Silicon in a physiological electrolyte wherein the bioactivity or biocompatibility of the porous silicon does not method comprises the application of an electrical bias to the arise. Silicon. The Silicon may be porous Silicon. Porous Silicon has been Suggested as a Substrate material In a further aspect, the invention provides bioactive for in vitro biosensors by M. Thust et al. in Meas. Sci. material characterised in that the bioactivity of the material Technol, Volume 7 1996 pages 26-29. In the device struc is controllable by the application of an electrical bias to the ture described therein, the porous Silicon is Subjected to a material. thermal oxidation process to form a Silicon dioxide layer on Conventional bioactive ceramics are electrically insulat the exposed Silicon Surfaces of the pores. Since the porous ing and therefore preclude their use in electrochemical Silicon is partially thermally oxidised, the bioactivity or 35 applications. Where the electrical Stimulation of tissue biocompatibility of the Silicon is not of relevance Since it is growth has been studied previously, it has often been diffi only the Silicon dioxide which is exposed to test Solutions. cult to distinguish the direct effects of electric fields from The porous Silicon is effectively an inert host for enzyme those associated with an altered body chemistry near Solutions. implanted “bioinert' electrodes. Microperforated silicon membranes have been described 40 In a Still further aspect, the invention provides a com as being capable of Supporting cell Structures by E. Richter posite Structure comprising bioactive Silicon region and a et al. in Journal of Materials Science: Materials in Medicine, mineral deposit thereon characterized in that the Silicon Volume 7, 1996, pages 85-97, and by G. Fuhr et al. in region comprises Silicon which is at least partly crystalline. Journal of Micromechanics and Microengineering, Volume A possible application of the invention is as a Substrate 5, Number 2, 1995, pages 77-85. The silicon membranes 45 for performing bioassays. It is desirable to be able to described therein comprises Silicon membranes of thickneSS perform certain tests on pharmaceutical compounds without 3 um perforated by Square pores of width 5 um to 20 um resorting to performing tests on living animals. There has using a lithography process. Mouse embryo fibroblasts were therefore been a considerable amount of research activity able to grow on cleaned membranes but adherence of the devoted to developing in vitro tests in which cell lines are cells was improved if the membranes were coated with 50 Supported on a Substrate and the effects of pharmaceutical polylysine. This paper is Silent as to the bioactivity of the compounds on the cell lines monitored. A composite Struc Silicon membrane, and there is no mention of an apatite layer ture of Silicon and apatite might provide a Suitable Substrate having been formed when exposed to the cell culture for Such tests. medium. Indeed, given the dimensions of the pores used, the In a further aspect, the invention provides a method of Structure is not likely to exhibit a Significant degree of 55 fabricating a biosensor, characterized in that the method bioactivity. Furthermore, it is accepted by Fuhr et al. that includes the Step of forming a composite Structure of bio there is still a need to find and develop cell-compatible active Silicon and a mineral deposit thereon. materials with long term Stability. The invention further provides a biosensor for testing the A.Offenhausser et al. in Journal of Vacuum Science pharmacological activity of compounds including a Silicon Technology A, Volume 13, Number 5, 1995, pages 60 Substrate, characterized in that at least part of the Silicon 2606-2612 describe techniques for achieving biocompat Substrate is comprised of bioactive Silicon. ibility with silicon substrates by coating the substrate with an ultrathin polymer film. Similarly, R. S. Potember et al. in BRIEF DESCRIPTION OF THE DRAWINGS Proc. 16th Int. Conf. IEEE Engineering in Medicine and In order that the invention may be more fully understood, Biology Society, Volume 2, 1994, pages 842-843 describe 65 embodiments thereof will now be described, by way of the use of a Synthetic peptide attached to a Silicon Surface to example only, with reference to the accompanying drawings, promote the development of rat neurons. in which: US 6,322,895 B1 S 6 FIG. 1 is a Schematic Sectional diagram of a bioactive hydrated porous Silicon wafers. The porous Silicon thus Silicon wafer; comprised a Silicon Skeleton coated in a thin native oxide, FIG. 2 is a representation of a Scanning electron micro Similar to that formed on bulk silicon as a result of Storage Scope (SEM) micrograph of an apatite deposit on a bulk in air. Silicon region adjacent a porous region of the FIG. 1 wafer; FIG. 3 is a representation of an SEM micrograph of a TABLE 1. croSS-Section of the FIG. 2 Silicon region; Concentration (mM FIG. 4 is a representation of an SEM micrograph showing an apatite Spherulite deposited on a porous Silicon region of Ion Simulated Body Fluid Human Plasma porosity 31% Nat 142.O 142.O K 5.0 5.0 FIG. 5a is a representation of an SEM micrograph of an Mg2+ 1.5 1.5 unanodised region of a Silicon wafer anodised to produce a Ca2+ 2.5 2.5 porosity of 48% after immersion in a simulated body fluid HCO, 4.2 27.0 Solution; 15 HPO2- 1.O 1.O C 147.8 103.0 FIG. 5b is a representation of an SEM micrograph of an SO, 0.5 0.5 anodised region of the FIG. 5a wafer; FIG. 6 is a Schematic diagram of a biosensor incorporat Cleaved wafer Segments having typical dimensions of ing bioactive Silicon; 0.4x50x20 mm were placed in 30 cm capacity polyethyl FIG. 7 is a Schematic diagram of an electrochemical cell ene bottles filled with the SBF solution and held at 37-1 for the electrical control of bioactivity; C. by a calibrated water bath. FIG. 8 is a plot of a calcium concentration profile in After a known period of time, the Segments were removed porous silicon wafers after treatment in the FIG. 7 cell; and from the SBF Solution, rinsed in deionised water and FIG. 9 is a schematic diagram of a biosensor device 25 allowed to dry in ambient air prior to characterisation. The incorporating bioactive polycrystaline Silicon of the inven SBF treated Segments were examined using Scanning elec tion. tron microscopy (SEM) and X-ray microanalysis (EDX) on a JEOL 6400F microscope. Secondary ion mass spectrom DETAILED DESCRIPTION OF THE etry was carried out using a Cameca 4F instrument and INVENTION infrared spectroscopy was performed using a Biorad FTS-40 Referring to FIG. 1 there is shown a section of a bioactive Spectrometer. silicon wafer, indicated generally by 10. The silicon wafer After periods of immersion in the SBF solution of 2, 4, 10 comprises a porous Silicon region 20 and a non-porous and 17 hours, there were negligible apatite deposits on both bulk Silicon region 22. The porous region 20 has a thickneSS the porous Silicon region 20 and the non-porous bulk silicon d of 13.7 um and an average porosity of 18%. The silicon 35 region 22. wafer 10 has a diameter 1 of three inches or 75 mm. The Referring to FIG. 2 there is shown a reproduction of an porous region 20 has a Surface area per unit mass of material SEM micrograph indicated generally by 50. The micrograph of 67 mg. This was measured using a BET gas analysis 50 is an image of part of the region 22 after the wafer 10 had technique, as described in "Adsorption, Surface Area and been placed in the SBF solution for a period of 6 days. A Porosity” by S. J. Gregg and K. S. W. Sing, 2nd edition, 40 Scale bar 52 indicates a dimension of 2 um. The micrograph Academic Press, 1982. 50 shows a continuous layer of apatite spherulites 54 cov The wafer 10 was fabricated by the anodisation of a ering the Surface of the region 22. The apatite spherulites had heavily arsenic doped Czochralski-grown (CZ) n-type (100) nucleated at a Sufficiently high density to create a relatively silicon wafer having an initial resistivity of 0.012 G2cm. The smooth film in which boundaries between spherulites such anodisation was carried out in an electrochemical cell, as 45 as boundary 56 are indistinct. The film was continuous over described in U.S. Pat. No. 5,348,618, containing an electro an area of at least 100 um. lyte of 50 wt % aqueous HF. The wafer was anodised using Referring to FIG. 3 there is shown a reproduction of an an anodisation current density of 100 mAcm for one SEM micrograph, indicated generally by 100, of a cross minute. The wafer was held in place in the electrochemical section of the wafer 10 in the region 22 after the wafer had cell by a synthetic rubber washer around the outside of the 50 been immersed in the SBF solution for 6 days. A scale bar wafer. Consequently, an outer ring of the wafer remained 102 indicates a dimension of 1.0 lum. The micrograph 100 unanodised after the anodisation process. This outer unano indicates three distinct regions, indicated by the letters A, B, dised. ring is shown in FIG. 1 as a non-porous bulk silicon and C. EDX analysis confirmed that region A is Silicon, region 22. The unanodised ring has a width S of 4 mm. corresponding to the original material of the non-porous In order to determine the bioactivity of anodised wafers, 55 bulk silicon region 22. Region B exhibited both silicon and cleaved wafer Segments were placed in a simulated body oxygen peaks under EDX analysis, indicating that region B fluid (SBF) solution for a period of time ranging from 2 comprises Silicon oxide. Region C exhibited calcium, phos hours to 6 weeks. The SBF solution was prepared by phorus and oxygen peaks under EDX analysis, consistent dissolving reagent grade Salts in deionised water. The Solu with this region comprising spherulites of apatite. The tion contained ion concentrations Similar to those found in 60 combined SEM and EDX analysis demonstrates that a human blood plasma. The SBF solution ion concentrations porous Silicon oxide layer (region B) has formed on the bulk and those of human blood plasma are shown at Table 1. The Silicon (region A), thereby enabling nucleation and coverage SBF solution was organically buffered at a pH of 7.30+0.05, with apatite (region C). SEM analysis of the wafer 10 in the equivalent to the physiological pH, with trihydroxymethy area of the porous Silicon region 20 after 6 days immersion laminomethane and hydrochloric acid. The porous wafers 65 in the SBF solution indicated a much lower level of apatite were Stored in ambient air for at least Several months prior coverage compared with the region 22. The porous Silicon to immersion in the SBF Solution and were therefore region 20 contains a high level of mesoporosity. After 10 US 6,322,895 B1 7 8 days immersion in the SBF solution in which significant despite being hydrated by exposure to ambient air, the layer erosion of the porous Silicon had occurred, macropores porous Silicon region maintains a high concentration of were visible under SEM analysis in the region 20. The quantum wires or dots. The luminescent property was pre combined SEM and EDX analysis demonstrates that, in served both during and after immersion in the SBF solution. contrast to the bulk silicon region 22, apatite nucleation can This shows that apatite may be deposited on porous Silicon occur directly on the porous Silicon region 20 and does not Such that the luminescent properties are preserved. Preser require the formation of an intermediate porous Silicon oxide Vation of the luminescent properties after growth of an layer. The intentional introduction of very large (greater than apatite layer may be a useful property for the development 100 um diameter) macropores may be advantageous in that of an electro-optical biosensor. it may enable vascular tissue to grow within the Structure of A wholly mesoporous luminescent porous Silicon wafer the porous Silicon. having a 1 um thick porous region with a porosity of 70% The formation of apatite deposits has also been observed and a Surface area per unit mass of 640 mg was placed on waferS having porous Silicon porosities other than 18%. in the SBF solution. After approximately one day the porous A microporous wafer having a porous Silicon region with a region had been completely removed by dissolution in the porosity of 31% was fabricated from a 0.03 G2cm heavily 15 SBF solution and the wafer was no longer luminescent. No boron doped p-type CZ Silicon wafer by anodisation at an apatite deposits were observed on either the porous Silicon anodisation current density of 100 mAcm' for one minute region or the non-porous region. It is thought that the in 50 wt % HF. The resulting porous silicon region had a mesoporous silicon is wetted more efficiently by the SBF thickness of 9.4 um and a Surface area per unit mass of 250 Solution and hence the rate of dissolution is higher for mig'. The porous silicon wafer was heavily aged prior to mesoporous Silicon than microporous Silicon. The mesopo immersion in the SBF solution. rous Silicon thus shows resorbable biomaterial characteris FIG. 4 shows a representation of an SEM micrograph, tics. It might be possible to construct a bioactive Silicon indicated generally by 150, of the surface of the 31% Structure having a limited area of mesoporous Silicon to act porosity porous Silicon layer after a Segment of the wafer as a Source of Soluble Silicon. This could produce a locally had been immersed in 30 cm of the SBF solution for 7 days. 25 Saturated Silicon Solution and hence the promotion of apatite The micrograph 150 shows spherulites such as a spherulite deposition. 152 of apatite on the Surface 154 of the porous silicon. A macroporous Silicon wafer having a porous region of Microporous waferS having a porous Silicon region of a 4% porosity and a thickness of 38 um behaved like a bulk, porosity of 48% were fabricated by anodising a lightly boron unanodised Silicon wafer in as much as it did not exhibit doped p-type Silicon wafer having a resistivity of 30 S2cm in growth of an apatite deposit when immersed in the SBF 50 wt % HF at an anodisation current density of 20 mAcmf Solution for four weeks. In addition, no apatite growth has for five minutes. The resulting porous Silicon region had a been observed on a porous Silicon region having a porosity thickness of 6.65 um and a Surface area per unit mass of of 80% and a thickness of 50 um which retains its lumines approximately 800 mg. The porous silicon wafer segment cent properties after two weeks immersion in the SBF was heavily aged prior to immersion in a 150 cm polyeth 35 Solution. ylene bottle filled with the SBF solution. AS a further control, a cleaved non-porous Silicon wafer FIG. 5a shows a representation of a SEM micrograph, Segment of Similar dimensions to the porous Silicon wafer indicated generally by 200, of an apatite deposit 202 on an segments was placed in 30 cm of the SBF solution. An unanodised region of the 48% porosity wafer after a four 40 extremely low density of micron size deposits, less than week immersion period. FIG. 5b shows a representation of 5000/cm’ was observed after immersion in the SBF solution a SEM micrograph, indicated generally by 250 of an apatite for five weeks. These deposits were possibly located at spherulite 252 deposited on the 48% porosity porous region. Surface defects of the Silicon wafer. Bulk, non-porous Silicon The spherulite 252 exhibits a morphology having a colum is therefore not bioactive since the rate of growth of apatite nar Structure characteristic of apatite growth on bioactive 45 deposits is too low for a bond to be formed with living tissue. ceramics as described by P. Liet al. in Journal of Biomedical These experiments thus indicate that by appropriate con Materials Research, Volume 28, pages 7-15, 1994. Apatite trol of pore size and porosity, Silicon Structures can cover Spherulites having a similar morphology were observed on virtually the entire bioactivity spectrum. Bulk and purely the unanodised region of the wafer. Cross-sectional EDX macroporous Silicon are relatively bioinert, high porosity spectra of the 48% porosity wafer after immersion in the 50 mesoporous Silicon is resorbable and microporous Silicon of SBF Solution taken acroSS the unanodised region indicated moderate porosity is bioactive. that Spherulites contained calcium, phosphorus and oxygen, It is known that changes in chemical composition of consistent with apatite. Away from the Spherulites, an inter biomaterials can also affect whether they are bioinert, facial layer having a thickness of only 150 nm comprising resorbable or bioactive. The above experiments were carried predominantly Silicon and oxygen was observed. Fourier 55 out on porous Silicon wafers which had not been intention transform infrared spectroscopy confirms the presence of ally doped with any specific elements other than the impu apatite in both the porous and non-porous regions. Both the rity doping for controlling the Semiconductor properties of P-O bending vibrational modes of PO tetrahedra at wave the Silicon. numbers of around 600 cm and a broadband around 1400 The elution of calcium from bioactive glass containing cm', attributed to vibrational modes of carbonate groups, 60 SiO, Na-O, CaO and POs is believed to significantly assist were observed. apatite growth by promoting local SuperSaturation. Calcium Some forms of porous Silicon are known to be photolu has been impregnated into a freshly etched layer of minescent. The observation of red or orange photolumines microporous Silicon of 55% porosity and having a thickness cence from porous Silicon generally indicates the presence of 1.2 um formed in a lightly doped p-type (30 S2cm) CZ of quantum wires or quantum dots of Silicon material. Prior 65 silicon wafer by anodisation at 20 mAcm for one minute to immersion in the SBF solution, the heavily aged 48% in 40% aqueous HF. The calcium impregnation was porosity wafer exhibited photoluminescence, indicating that achieved through mild oxidation by Storage in a Solution US 6,322,895 B1 9 10 containing 5 g of CaCl2.H2O in 125 cm pure ethanol for 16 a porous Silicon region in order to promote the deposition of hours. The impregnation of the porous Silicon with calcium, apatite and the bonding of the sensor with the tissue. In FIG. Sodium or phosphorus or a combination of these species may 6, the porous silicon is indicated by rings 330 on the top promote apatite formation on Silicon. surface of the segment 304 and grooves 332 in the other The presence of the Silicon oxide layer underneath the surfaces. Although FIG. 6 indicates that the outer surfaces of apatite deposit at the non-porous region adjacent the porous the segments 302 and 304 are covered entirely by porous Silicon region of the anodised wafers after immersion in the silicon, it may be sufficient for only the Surface 322 and a SBF solution indicates that the dissolution of silicon from bottom surface 334 of the segment 302 to incorporate porous the porous Silicon region may be an important factor for the Silicon. Such an arrangement would be simpler to fabricate. bioactivity of the porous silicon. The dissolution of the The segments 302 and 304 are bonded together using Silicon may form a local SuperSaturated Solution which techniques developed for Silicon on insulator technologies. results in the deposition of a porous Silicon oxide layer. Whilst an anodisation technique has been described for the Apatite is then deposited on the porous Silicon oxide. This production of the porous Silicon, Stain etching techniques are Suggests that a variety of non-porous crystalline, polycrys also known for the production of porous Silicon. Such talline or amorphous Silicon based Structures containing 15 techniques may be advantageous for producing porous Sili impregnated calcium and having a higher Solubility than con Surfaces on complex shaped Structures. normal bulk crystalline silicon in the SBF solution may be In addition to Sensors, bioactive Silicon might find appli bioactive. To Significantly assist apatite growth, the level of cations in electronic prosthetic devices, for example replace calcium impregnation needs to be much higher than previ ment eyes. Other electronic devices which may incorporate ously reported calcium doped Silicon, though the crystallin bioactive Silicon might include intelligent drug delivery ity of the Silicon need not necessarily be preserved. Systems. Calcium is generally regarded as an unattractive dopant AS well as Sensors for incorporation into the bodies of for Silicon and consequently there have been few Studies of humans and other animals, bioactive porous Silicon may be calcium doped Silicon. Sigmund in the Journal of the Elec used in the fabrication of biosensors for in vitro applications. 25 A composite Structure of porous Silicon with a layer of trochemical Society, Volume 129, 1982, pages 2809 to 2812, apatite thereon may have improved cell compatibility com reports that the maximum equilibrium Solubility of calcium pared with prior art biosensor arrangements. BioSensors are in monocrystalline silicon is 6.0x10 cm. At this of potentially great importance in the field of in Vitro concentration, calcium is unlikely to have any significant pharmaceutical testing. For automated pharmaceutical effect upon apatite growth. SuperSaturated levels of calcium testing, a bioasay device might comprise a Silicon wafer are needed with concentrations in excess of 10° cm (2 at having a matrix array of porous Silicon regions. Cells could %). Such very high concentrations may be achieved by: then be preferentially located at the porous Silicon regions (a) Solution doping of porous Silicon as previously and this would facilitate automated cell analysis after expo described; Sure to a pharmaceutical product. The luminescent proper (b) ion implantation of porous Silicon or bulk silicon with 35 ties of porous Silicon might be utilised to enable an optical calcium ions, or cell analysis technique. Workers skilled in the field of (c) epitaxial deposition of calcium or calcium compounds biosensors would use their experience to identify which cell followed by thermal treatments. cultures were Suitable and how the cells behavior could be Referring to FIG. 6 there is shown a schematic diagram of monitored. a generalised Sensor, indicated generally by 300, for medical 40 Whilst the results of in vitro experiments have been applications incorporating bioactive silicon. The sensor 300 described, no in Vivo experiments have been described. comprises two silicon wafer segments 302 and 304. The However, the in Vitro experiments are designed to mimic the segment 302 incorporates CMOS circuitry 306 and a sensing environment within a human body. From the results of the element 308 linked to the circuitry 306. The sensing element in vitro experiments it may be concluded that those Silicon 308 may be an oxygen sensor, for instance a Clark cell. The 45 wafers which produced significant deposits of apatite in the CMOS circuitry is powered by a miniaturised battery (not SBF Solution would also exhibit bioactive behavior in vivo. shown) and signals are produced for external monitoring The formation of a film of apatite over a Silicon or porous using Standard telemetry techniques. Silicon Surface in vitro indicates that the bioactive Silicon The wafer segment 304 is a micromachined top cover for may be to a certain extent a biocompatible form of Silicon. the segment 302. The segment 304 has two major cavities 50 The term “biocompabble' does not necessarily indicate that 310 and 312 machined into it. The cavity 310 has a dome the material is biologically acceptable for all applications shape. When the segments 302 and 304 are joined together, but that the material is biologically acceptable for Specific the cavity 310 is above the CMOS circuitry 306. The cavity applications. Some workers skilled in the field of biocom 312 is circular in croSS-Section and extends through the patibility might regard “tissue compatible' as a more appro segment 304 to allow the sensing element 308 to monitor the 55 priate term to describe this definition of biocompatibility. environment Surrounding the Sensor. The cavity 312 is The layer of apatite may act as a protective barrier reducing covered by a permeable membrane 314. In addition to the the physiological effects of the Silicon. major cavities 310 and 312, minor cavities, such as cavities AS Stated above, mesoporous Silicon shows resorbable 316, are distributed over a top surface 322 of the segment biomaterial characteristics. From the previously referenced 304. The minor cavities are frusto-conical in shape, with the 60 paper by Hench in the Journal of the American Ceramic diameter of its cross-section increasing into the Segment. Society, resorbable biomaterials are materials which are The minor cavities are present to enable the growth of designed to degrade gradually over a period of time and be vascular tissue or bone for biological fixation. The cavities replaced by the natural host tissue. The characteristics of the 310, 312, and 316 are formed by standard etching mesoporous Silicon in the Simulated body fluid indicate that techniques, for example ion-beam milling and reactive ion 65 mesoporous Silicon of an appropriate porosity may be a etching through a photoresist mask. At least part of the outer resorbable biomaterial. AS previously discussed the porous surfaces of the segments 302 and 304 are anodised to form region 20 of the bioactive silicon wafer 10 of FIG. 1 contains US 6,322,895 B1 11 12 a high level of mesoporosity. This indicates that controlling rous containing mineral, with other SBF constituents Such as the porosity of mesoporous Silicon can control whether a carbon, magnesium, Sodium and chlorine being close to porous Silicon region is bioactive or resorbable. It may be EDX detection limits (i.e. <1 atomic %). Plan view EDX possible to control the rate at which a porous Silicon region analysis of the unbiased and anodically biased wafers is absorbed by tuning the porosity. showed only the presence of Silicon and oxygen. Although the dissolution of porous silicon in the SBF Cross-sectional SEM and EDX analysis showed that the Solution provides an indication of resorbable biomaterial calcium and phosphorous rich mineral developed under characteristics, the behavior of a porous Silicon region in a cathodic bias is restricted to the top of the porous Silicon living body may be affected by factors which are not layer and is relatively thin, having a thickness of approxi reproducible in the SBF solution. If living cells grow on the mately 0.2 um. Within the porous silicon the calcium and Surface of the porous Silicon, these cells may interact with phosphorous levels are below EDX detection limits for all the porous silicon. Thus experiments carried out in the SBF Samples. The porous Silicon layer given the anodic loading solution do not provide a clear indication of the suitability of showed a Significant build up of oxygen within the top 0.5 a particular form of porous Silicon for resorbable material tim of the layer. applications. Experiments may have to be carried out in vivo 15 Secondary ion mass spectrometry (SIMS) was utilised to to determine whether a particular desired physiological compare the extent and depth to which layers were calcified response is achieved. after the three differing treatments, together with the depth Further experiments have been performed which show distribution of other specific elements. Freshly etched that it is possible to either enhance or retard the formation of microporous Silicon has been shown to contain very low an apatite layer on the porous Silicon by the application of levels of for example calcium and sodium (present in SBF) a bias current in the SBF solution. but appreciable levels of fluorine (not present in SBF). Referring to FIG.7 there is shown a schematic diagram of FIG. 8 is a SIMS plot shows the varying levels of an electrochemical cell 400 for applying a galvanoStatic calcification resulting from the electrical biasing treatments. loading to a whole silicon wafer 402. The wafer 402 is a In FIG. 8, the SIMS plot from a cathodically biased wafer is heavily doped n-type (100) oriented silicon wafer of resis 25 shown by a line 450, the SIMS plot from an unbiased wafer tivity 0.012 S.2cm which prior to loading in the cell 400 was is shown by line 452, and a SIMS plot from an anodically anodized in 40 wt % aqueous HF at 100 mA cm for one biased wafer is shown by a line 454. Although deposition minute to form a bioactive porous Silicon layer of approxi has primarily occurred near the Surface of the porous Silicon, mately 20% porosity having a thickness of 11 um with a in all cases calcium levels were above the background level BET measured surface area of approximately 70 mig". throughout the 11 uA thick layer. The line 450 shows that After anodisation, the wafers are spun dry in air until their cathodic biasing has raised the degree of calcification and weight has Stabilised and then immediately loaded into the anodic biasing has lowered it compared with the unbiased cell 400. wafer. The SIMS measurements also indicated the presence The wafer 402 is inserted into a PTFE cassette 404 and of the SBF constituents throughout the porous silicon layer mounted using a threaded PTFE ring 406 which is screwed 35 and that there had been Significant movement and loSS of into the cassette 404 and which compress PTFE coated fluorine as a result of the cathodic biasing, together with O-rings 408 and 410. In the cassette 404, the silicon wafer Some degree of retention within the overlayer. is pushed against a metal back plate 412. The plate 412 It is well established that in vitro and in vivo tissues only provides an electrical contact to a rear face of the Silicon respond favorably over quite restricted ranges of input wafer, and in the cassette an area of 36 cm of the front 40 power, current and Voltage in electroStimulation experi porous face of the silicon wafer is exposed. The cassette 404 ments. These ranges are Sensitive to many factors including is placed in a polycarbonate tank 414, within a waterbath, the nature of the Stimulating electrodes. The biasing experi containing two litres of SBF solution maintained at 37+1 C. ments described above indicate that the kinetics of the with organic buffering at pH=7.3+0.05. A spiral platinum calcification process of porous Silicon can be accelerated in counterelectrode 416 is also inserted into the SBF solution. 45 vitro and therefore possibly in vivo by the application of a A d.c. galvanostatic power Supply 418 is used to maintain a cathodic bias. They also Suggest that when dissimilar Silicon constant electrical current between the wafer 402 and the Structures Such as porous and bulk Silicon are immersed counterelectrode 416. The wafer 402 may either be under together in physiological electrolytes, galvanic corrosion cathodic or anodic bias control. The power Supply 418 processes may favour calcification at any cathodic Sites that provides a constant current of 36 mA, which corresponds to 50 develop. a current density. at the Silicon wafer of approximately 1 mA The potential applications for the bias control of mineral cm° if current flow is primarily through the silicon skeleton deposition are varied. It is known that the insertion of or approximately 1 uA cm if current flow is uniformly electrodes into a living organism may result in the formation distributed across the entire silicon-SBF interface via the of a fibrous layer around the electrode, with the thickness of pore network of the porous silicon. The current flow is 55 the layer being an indication of the biocompatibility of the maintained for three hours. After removal from the cell 400, electrode. The rapid formation of a Stable mineral deposit the wafers 402 are rinsed in deionised water and spun dried. around microelectrodes in vivo offers potential benefits for After the three hour SBF exposure, the porous silicon the electroStimulation of tissue growth or the Stimulation of wafer Surface was examined in a JEOL 6400F scanning muscles of paraplegics. The localised control of mineral electron microscope (SEM) at an accelerating potential of 6 60 deposition, where localised regions may be arranged So that kV. Porosified wafers which were anodically biased, a mineral deposit is not formed thereon might have appli together with control porosified wafers which received no cations in the field of biosensing devices, both in Vivo and bias showed no evidence of Surface deposits on the porous in vitro. The process of enhanced mineral deposition may be silicon. The wafer which was cathodically biased however beneficial in the coating of Silicon based integrated circuits was completely covered with Spherulites which had merged 65 prior to their implantation in the body. to form a continuous layer. Plan view EDX analysis showed Whilst the above description of the electrical control of that this overlayer is a predominantly calcium and phospho the deposition of a mineral is concerned with the deposition US 6,322,895 B1 13 14 on porous Silicon, mineral deposits have also been observed the deposits appearing to be more angular. The reasons for when a cathodic bias is applied to an unanodised wafer in the this are not understood but could reflect a slightly different SBF Solution. local pH at the nucleation sites on the polysilicon. P. Li et al. In a further embodiment, it has been found that certain in Journal of Applied Biomaterials, Volume 4, 1993, page types of polycrystalline Silicon (polysilicon) are also capable 221, reported that the apatite morphology observed at a pH of inducing calcium phosphate deposition from an SBF of 7.3 is significantly different from that observed at a pH of Solution and are hence bioactive. 7.2 for growth on Silica gel. In order to produce bioactive polycrystalline silicon, 100 The potential applications for bioactive polysilicon are mm diameter <100>p-type CZ silicon wafers having a resistivity in the range 5 to 10 S2cm are coated front and back potentially broader than those for bioactive porous Silicon. It with a 0.5 um thick wet thermal oxide and Subsequently a 1 is possible to coat a variety of Substrates with polysilicon tim thick polysilicon layer of varying microstructure. The which could not be coated with monocrystalline Silicon. oxide layer is grown in a Thermco TMX9000 diffusion Surgical implants could be coated with a layer of polysilicon furnace and the polysilicon layer is grown in a Thermco in order to improve adhesion with bone. Polysilicon is also TMX9000 low pressure chemical vapour deposition hot 15 highly compatible with VLSI technology offering the pros walled furnace. For thermal oxide growth, the furnace tube pect of complex electronic circuitry being made biocompat is held at a uniform temperature of 1000 C., and the wet ible. Polysilicon can be surface micromachined in order to thermal oxide is grown using Steam oxidation for 110 produce a variety of devices and packaging arrangements. minutes. The Subsequent deposition of the polysilicon layer One possible bioactive Silicon packaging concept has involves the pyrolysis of SiH at a pressure in the range 250 already been described with reference to FIG. 6. With to 300 mtorr with the furnace tube held at a temperature in bioactive polysilicon, it might be possible to construct the range 570 to 620° C. smaller biochips. Referring to FIG. 9 there is shown a It is well established that the microstructure of the poly Schematic diagram of a biosensor device 500 incorporating Silicon layer is Sensitive to many deposition parameterS Such bioactive polysilicon. The device 500 comprises a bulk as temperature, pressure, gas flow rate, and Substrate type, as 25 Silicon wafer 510 onto which a CMOS circuit 512 and a described in Chapter 2 of “Polycrystalline Silicon for Inte sensor element 514 are fabricated. The sensor element 514 grated Circuit Applications” by T. Kamins, published by is electrically connected to the circuit 512. The circuit 512 Kluwer Acad. Publ. 1988. Polysilicon layers of widely is protected by a barrier layer 516 of for example silicon varying microstructure and morphology were obtained by oxide and silicon nitride. The whole of the device 500 except using different deposition temperatures of 570° C., 580 C., for a window 518 to the sensor element 514 is covered with 590° C., 600° C., 610° C. and 620 C. Cross-sectional a layer 520 of bioactive polysilicon. The barrier layer 516 is transmission electron microScopy analysis revealed that the required because polysilicon itself is not a good protective layer deposited at 570 C. was virtually amorphous near its layer for Silicon based circuitry due to diffusion through surface whereas the layers deposited at 600 C. and 620 C. grain boundaries. The barrier layer 516 is therefore inter were polycrystalline throughout their depths. The grain size 35 posed between the circuit 512 and the polysilicon layer 520. varies appreciably with deposition temperature and Signifi By analogy with the results using porous Silicon, the cantly with depth for a given layer. bioactivity of polycrystalline silicon might be improved by Cleaved wafer Segments having typical dimensions of doping it with calcium, Sodium or phosphorus or a combi 0.5x50x20 mm were then placed in separate 30 cm nation of these Species. polyethylene bottles filled with SBF solution as previously 40 Bioactive polysilicon might be a suitable substrate for described, with the temperature of the SBF maintained at bioassay device applications. L. Bousse et al. in IEEE 37 C.1 C. The different polysilicon layers were observed Engineering in Medicine and Biology, 1994 pages 396 to to have varying levels of stability in the SBF solution as 401 describe a biosensor for performing in Vitro measure determined by cross-sectional SEM imaging. After 64 hours ments in which cells are trapped in micromachined cavities in the SBF solution, the polysilicon layer deposited at 620 45 on a Silicon chip. Such an arrangement might beneficially C. was thinned to approximately 60% of its original incorporate a composite Structure of polysilicon with a layer thickness, whereas the thickness of the layer deposited at of apatite thereon, the cells locating themselves preferen 570° C. was substantially unchanged after 160 hours in the tially on regions of apatite. SBF Solution. What is claimed is: Mineral deposits were observed to nucleate and prolifer 50 1. A composite Structure comprising a bioactive Silicon ate over certain of the polysilicon layers. These deposits region and a mineral deposit thereon. were observed using plan-view SEM. After two weeks 2. A composite Structure according to claim 1, wherein the immersion in the SBF solution, mineral deposits were mineral deposit is apatite. observed on the polysilicon layers deposited at 600 C. and 3. A composite Structure according to claim 1 or claim 2, 620° C. but not on the layer deposited at 570° C. These 55 wherein the bioactive Silicon region is porous Silicon. observations indicate that as for the porous Silicon there is a 4. A composite Structure according to claim 1 or claim 2, reactivity window, dependent on the microStructure, for wherein the bioactive Silicon is polycrystalline Silicon. optimum bioactivity. The greatest density of mineral depos 5. A composite Structure according to 1 wherein the its were observed with the polysilicon layer deposited at bioactive Silicon comprises Silicon Selected from the group 600 C. Significant levels of mineral deposits were observed 60 consisting of porous crystalline Silicon, porous amorphous on both the front and back of the silicon wafers, consistent Silicon, porous polycrystalline Silicon, non-porous crystal with there having been polysilicon deposition on both Sides. line Silicon, and non-porous polycrystalline Silicon. EDAX analysis of the deposits indicated the presence of 6. A method of fabricating a biosensor, wherein the calcium, phosphorous and oxygen, consistent with Some method includes the Step of forming a composite Structure of form of apatite having nucleated. The morphology of the 65 bioactive Silicon and a mineral deposit thereon. deposits however differs from that of the spherulites previ ously described in connection with the porous Silicon, with